Our long-term goal is to understand how visual information is processed by the retina at the level of specific cell types and synapses. Retinal processing depends critically on a vertical, excitatory glutamatergic pathway, from photoreceptors bipolar cells ganglion cells (output neurons). Bipolar cell synapses have been proposed to express multiple important functions, including: the ability to release at high rates to maintain a high signal-to-noise ratio; the ability to release either synaptically or extrasynaptically and thereby signal different receptor populations; the ability to generate visual adaptation through use-dependent plasticity; and the ability to generate receptive field properties, including direction selectivity. All proposed functions have been difficult to study wih the common method of whole-cell patch clamp recording: patch recording cannot resolve individual synaptic inputs within the dendritic tree and cannot accurately measure large conductances or small signals on distal dendrites. Here, we propose a new combination of methods that will yield fundamental insights into bipolar cell synaptic transmission. We will use a newly-developed genetically encoded glutamate sensor (iGluSnFR), delivered with a virus injected into the vitreous of the eye and expressed under the control of promoters that drive expression in identified postsynaptic targets of bipolar cells. When iGluSnFR binds glutamate, its fluorescent properties change and these changes can be measured with two-photon microscopy. We have a working two-photon microscope system that is optimized for fluorescence measurements during cone photoreceptor stimulation in vitro. Aim 1 will characterize glutamate release onto amacrine and ganglion cells down to the level of individual synapses and vesicles. We will correlate iGluSnFR signals with spontaneous and light-evoked electrical events, in time, and with synaptic locations, in space. Glutamate spillover will be imaged under conditions with either elevated release or limited glutamate uptake. Aim 2 will image synaptic depression at individual synapses and test its role in contrast adaptation. Aim 3 will test for parallel processing at the output of single bipolar terminals by measuring directiona selectivity of glutamate release at individual synapses onto direction-selective ganglion cells. These studies will yield fundamental insights into the functional organization of the retina at the level of synapses and will be applicable to study the effects of aging and disease on retinal function.